Abstract With an estimated 1.5 million deaths in 2015, tuberculosis is the leading infectious killer worldwide, ahead of HIV/AIDS. Within this population are increasing numbers of tuberculosis strains resistant to standard courses of treatment. To prevent the spread of M. tuberculosis (Mtb) strains, new strategies for the rapid and accurate diagnosis of tuberculosis are needed. The current methods for direct detection of Mtb rely on old, lengthy procedures that are extensive, leading to varying degrees of sensitivity depending on method and clinician technique. To improve time-to-diagnosis and analysis accuracy, we have developed an environment-sensitive mycobacterial probe, termed DMN-Tre. 4-N,N-dimethylamino-1,8-napthalimide (DMN) is a fluorogenic dye that only fluoresces in hydrophobic environments. Conjugated to trehalose, we hypothesized this probe would allow fast and simple detection of mycobacteria. Preliminary data demonstrate that DMN-Tre quickly labels Mtb in a simple and fast protocol. More importantly, this compound is specific and selective for active Mtb, which can help stratify patients for treatment. In this proposal, we will characterize mycobacterial labeling with DMN-Tre in order to evaluate its performance for Mtb detection and validate specificity of incorporation in the mycombrane (Specific Aim1A). The ability to distinguish between live and dead bacteria suggests that it may be a powerful tool for studying how M. tuberculosis can respond to drug treatment. Thus, we will next use DMN-Tre to study the trehalose mycolate dynamics in response to TB drug treatments (Specific Aim1B). In Aim 2, we will investigate whether DMN-Tre is suitable for the fast, sensitive detection of Mtb in patient sputum samples. Using mock sputum samples with various amounts of Mtb, we will first determine the limit of detection of DMN-Tre. Given that Mtb reside in the host in an active and persistent state, we will investigate whether DMN-Tre can distinguish replicating and non-replicating mycobacteria (Specific Aim2A). Lastly, we will perform head-to-head comparison experiments to determine the sensitivity of DMN-Tre compared to current clinically used Mtb stains for TB diagnosis (Specific Aim2B). If higher, the impact of this work may lead to a complete modernization of the current state of TB diagnosis in high-burden regions.